The present invention, in some embodiments thereof, relates to methods of expanding mesenchymal stem cells and cell populations generated thereby.
Mesenchymal stem cells (MSCs) are non-hematopoietic cells that are capable of differentiating into specific types of mesenchymal or connective tissues including adipose, osseous, cartilaginous, elastic, neuronal, hepatic, pancreatic, muscular, and fibrous connective tissues. The specific differentiation pathway which these cells enter depends upon various influences from mechanical influences and/or endogenous bioactive factors, such as growth factors, cytokines, and/or local microenvironmental conditions established by host tissues.
MSCs reside in a diverse host of tissues throughout the adult organism and possess the ability to ‘regenerate’ cell types specific for these tissues. Examples of these tissues include adipose tissue, umbilical cord blood, periosteum, synovial membrane, muscle, dermis, pericytes, blood, bone marrow and trabecular bone.
The multipotent character of mesenchymal stem cells make these cells an attractive therapeutic tool and candidate for transplantation, capable of playing a role in a wide range of clinical applications in the context of both cell and gene therapy strategies. Mesenchymal cells may be used to enhance hematopoietic engraftment post-transplantation, to correct inherited and acquired disorders of bone and cartilage, for implantation of prosthetic devices in connective and skeletal tissue, and as vehicles for gene therapy.
In culture, expanded MSC express a panel of key markers including CD105 (endoglin, SH2), CD73 (ecto-5′ nucleotidase, SH3, SH4), CD166 (ALCAM), CD29 (β1-integrin), CD44 (H-CAM), and CD90 (Thy-1). In contrast to hematopoietic stem cells they lack CD45, CD34 and CD133 expression.
MSC can be identified by their ability to form colony forming units-fibroblast (CFU-F) in vitro. However, these cells are heterogeneous with respect to their proliferation and differentiation capacity. At least two morphologically distinct MSC populations have been identified that differ not only in size but also in their cell division rate and differentiation capacity. In addition, analysis of single cell-derived MSC colonies from adult bone marrow revealed differential capacity of colonies to undergo osteogenic, adipogenic, and chondrogenic differentiation.
In most cases, unfractionated bone marrow-derived cells are used as the starting population for the culture of MSC. This isolation method relies on the adherence of fibroblast-like cells to a plastic surface and the removal of non-adherent hematopoietic cells. The resulting cells are poorly defined and give rise not only to heterogeneous MSC populations but also to osteoblasts and/or osteoprogenitor cells, fat cells, reticular cells, macrophages, and endothelial cells. To define the starting population more precisely, surface markers such as SH2 (CD105), SH3/SH4 (CD73), SSEA-4 and the low affinity nerve growth factor receptor (CD271), which enrich for MSC, have been employed [Simmons P. J et al. (1991) Blood 78:55-62; Conconi M T et al., (2006) Int J Mol Med 18:1089-96; Gang E J et al., (2007) Blood 109:1743-51; Liu P G, (2005); Zhongguo Shi Yan Xue Ye Xue Za Zhi 13:656-9; Quirici N, et al., (2002) Exp. Hematol 30:783-91].
Another example of a cell surface antigen which has been targeting for isolating homogeneous populations of mesenchymal stem cells is stromal precursor antigen-1 (STRO-1). The STRO-1 antigen is expressed on the surface of approximately 10-20% of adult human BM that includes all CFU-F, Glycophorin-A nucleated red cells, and a small subset of CD19 B-cells, but is not expressed on hematopoietic stem and progenitor cells (HSC) (Simmons and Torok-Storb, 1991). STRO-1 is widely regarded as a marker of early mesenchymal/stromal precursor cells, because it has been strongly linked to mesenchymal cell clonogenicity, plasticity, and other progenitor cell characteristics [Psaltis et al., (2010), Journal of Cellular Physiology, 530-540]. High co-expression of STRO-1 (STRO-1Bright) with other surface markers, such as CD106, CD49a, CD146 or STRO-3 has been shown to greatly increase the cloning efficiency of BM MNC (Gronthos et al., 2008, Methods Molecular Biology, 449:45-57]. Freshly isolated STRO-1Bright BM MNC also possess other hallmark features characteristic of multipotent stem cells, including in vivo quiescence, high telomerase activity, and an undifferentiated phenotype. Moreover, this population of cells lacks hematopoietic stem cell (CD34), leukocyte (CD45), and erythroid (Glycophorin-A) associated markers.
More recently, platelet derived growth factor receptor-β (PDGF-RB; CD140b) was identified as a selective marker for the isolation of clonogenic MSC [Buhring H J, (2007) Ann N Y Acad Sci 1106:262-71]. Other reports demonstrated a 9.5-fold enrichment of MSC in bone marrow cells with prominent aldehyde dehydrogenase activity [Gentry T et al., (2007) Cytotherapy 9:259-74].
Even though MSCs multiply relatively easily in vitro, their proliferative potential and their stem cell characteristics are continuously decreased during prolonged culture. For example, it has been shown that expansion in culture leads to premature senescence (the process of aging characterized by continuous morphological and functional changes). Cells became much larger with irregular and flat shape and the cytoplasm became more granular. These senescence-associated effects are continuously acquired from the onset of in vitro culture (PLoS ONE, May 2008|Volume 3|Issue 5|e2213). As a result, the successful manufacturing for commercialization of large batches from one donor of homogenous MSCs that preserve their characteristics following expansion in culture remains a challenge.
Due to the low or absent expression of MHC molecules, especially class II on mesenchymal stem cells, these cells may be considered immune privileged, thus paving the way for allogeneic transplantation of such cells for the treatment of a wide range of disorders. Accordingly, improved methods of expanding banks of mesenchymal stem cells have become an important factor for commercializing their use.
The role of growth factors in increasing proliferation and survival in MSCs has been widely studied over the past few years and many factors have been proposed for increasing the expansion efficiency of these cells.
For example, many protocols relating to the expansion of MSCs include culturing in the presence of basic fibroblast growth factor (b-FGF) (Vet Res Commun. 2009 December; 33(8):811-21). It has been shown that b-FGF not only maintains MSC proliferation potential, it also retains osteogenic, adipogenic and chondrogenic differentiation potentials through the early mitogenic cycles.
Vascular endothelial growth factor (VEGF) has also been shown to increase MSC proliferation [Pons et al., Biochem Biophys Res Commun 2008, 376:419-422].
Exogenous addition of Hepatocyte growth factor (HGF) to MSC populations has been shown to affect proliferation, migration and differentiation (Furge et al., Oncogene 2000, 19:5582-5589].
Another proposed growth factor for increasing the expansion of MSCs is Platelet derived growth factor (PDGF) shown to be a potent mitogen of MSCs [Kang et al., J Cell Biochem 2005, 95:1135-1145].
Epidermal growth factor (EGF) and heparin-binding EGF have both been shown to promote ex vivo expansion of MSCs without triggering differentiation into any specific lineage [Tamama et al., Stem Cells 2006, 24:686-695; Krampera et al., Blood 2005, 106:59-66]. In addition to its mitogenic effect on MSCs, EGF also increases the number of colony-forming units by 25% [Tamama et al., J Biomed Biotechnol 2010, 795385].
Other have suggested the use of Wnt signalling agonists for expanding MSCs based on experiments which study Wnt signaling proliferation in MSCs. Canonical Wnt signalling was shown to maintain stem cells in an undifferentiated but self-renewing state. Addition of Wnt3a by activating the canonical Wnt pathway increased both proliferation and survival while preventing differentiation into the osteoblastic lineage in MSCs [Boland et al., J Cell Biochem 2004, 93:1210-1230].
The choice of growth factors to be used on MSCs was initially determined based on previously existing knowledge about the effect of a particular growth factor on cell morphogenesis. This was done with the dual pursuit of expanding MSCs and causing them to differentiate into the lineage that it was known to favor. Transforming growth factor beta (TGFβ), for example, is known to influence cells from the chondrogenic lineage in vivo, promoting initial stages of mesenchymal condensation, prechondrocyte proliferation, production of extracellular matrix and cartilage-specific molecule deposition, while inhibiting terminal differentiation. When applied to MSCs, cells show increased proliferation and a bias towards the chondrogenic lineage [Bonewald et al., J Cell Biochem 1994, 55:350-357; Longobardi L, J Bone Miner Res 2006, 21:626-636.
BMP-3, another member of the transforming growth factor beta family, known to enhance bone differentiation was shown to increase MSC proliferation threefold [Stewart A et al., Cell Physiol 2010, 223:658-666].
Nicotinamide (NA), the amide form of niacin (vitamin B3), is a base-exchange substrate and a potent inhibitor of NAD(+)-dependent enzymes endowed with mono- and poly-ADP-ribosyltransferase activities. ADP-ribosylation is implicated in the modification of a diverse array of biological processes (Corda D, Di Girolamo M. 2003; 22(9):1953-1958; Rankin P W, et al., J Biol Chem. 1989; 264:4312-4317; Banasik M. et al., J Biol Chem. 1992; 267:1569-1575; Ueda K, Hayaishi O, Annu Rev Biochem. 1985; 54:73-100; Smith S. Trends Biochem Sci. 2001; 26:174-179; Virág L, Szabó C. Pharm. Reviews. 2002; 54:375-429).
WO 07/063545 discloses the use of nicotinamide for the expansion of hematopoietic stem and/or progenitor cell populations.
WO 03/062369 discloses the use of nicotinamide, and other inhibitors of CD38, for the inhibition of differentiation in ex-vivo expanding stem and progenitor cells. However, WO 03/062369 does not teach administration of nicotinamide for particular time intervals.
U.S. Patent Application No. 20050260748 teaches isolation and expansion of mesenchymal stem cells with nicotinamide in the presence of a low calcium concentration.
Additional background art includes Farre et al., Growth Factors, 2007 April; 25(2):71-6.